Tumor suppressor gene del-27

ABSTRACT

The invention comprises an isolated nucleic acid molecule having SEQ ID NO:1 and the complementary sequence, as well as nucleic acid molecules hybridizes under stringent conditions to SEQ ID NO:1. SEQ ID NO:1 is a new tumor suppressor gene.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This is a continuation of copending patent Application Ser. No.09/255,947, filed Feb. 23, 1999.

FIELD OF INVENTION

[0002] The present invention relates to the new tumor suppressor genedel-27, the protein coded thereby, and their use for diagnostics andtherapeutics, especially in the field of cancer. In particular, theinvention relates to the diagnosis of the lack of tumor suppressor genedel-27 in mammalian, especially in tumor, cells and gene therapy methodsto restore del-27 and its functions in mammalian cells, especially intumor cells.

BACKGROUND OF THE INVENTION

[0003] Tumor suppressor genes are typically thought of as genes whoseexpression is reduced or lost in cancer cells (Knudson, Proc. Natl.Acad. Sci. USA 19 (1993) 10914-10921). The lack of expression resultsfrom mutations in the genes encoding their proteins. Since theseproteins are believed to suppress cell growth and thereby act asnegative growth regulators, loss of their expression in tumor cellsleads to the increased cell proliferation observed and contributes tomalignant transformation. As negative growth regulators, tumorsuppressor gene products are likely to have also normal functionscritical to the development of differentiated tissues. In this respect,tumor suppressor genes may have an important role in the growth arrestnecessary for the onset of cellular differentiation as growth regulationis a normal feature of development and differentiation. An overview oftumor suppressor genes is given by, e.g., Gutmann, D. H., Int. J. Dev.Biol. 39 (1995) 895-907.

[0004] Inactivation of tumor suppressor genes appears to be apredominant genetic event in the genesis and progression of many tumors.In normal cells, these genes are thought to be involved in theregulation of cell proliferation and differentiation (Fearon, E., andVogelstein, B., Cell 61 (1990) 759-767). Inactivating mutations anddeletions of tumor suppressor genes may therefore release normal growthconstraints and may result in the development or progression of tumorcells. A genomic region that contains a putative tumor suppressor genecan be identified by frequent loss of heterozygosity (LOH) of the normalallele with the remaining allele beeing presumably non-functional in thetumor cells (Fearon, E., and Vogelstein, B., Cell 61 (1990) 759-767).

[0005] Recently, frequent LOH at the del-27 locus on the short arm ofchromosome 5 in human lung carcinomas was demonstrated (Wieland, I., andBöhm, M., Cancer Res. 54 (1994) 1772-1774; Wieland, I., et al., Oncogene12 (1996) 97-102). Furthermore, LOH at the del-27 locus correlated withtumor progression in transitional cell carcinoma (B{umlaut over (o )}hm,M., Int. J. Cancer 72 (1997) 1-5). del-27 was isolated by genomicdifference cloning and is homozygously deleted in a lung carcinoma cellline supporting its close linkage to a novel putative tumor suppressorgene (Wieland, I., Proc. Natl. Acad. Sci. USA 87 (1992) 2720-2724).However, no cDNA coding for a tumor suppressor gene was describedtherein.

SUMMARY OF THE INVENTION

[0006] It is an object of the invention to provide a novel tumorsuppressor gene from the del-27 locus. The invention comprises anisolated nucleic acid molecule (del-27) having the nucleic acid sequenceSEQ ID NO:1. The invention also comprises an isolated nucleic acidmolecule that hybridizes under stringent conditions with each of nucleicacid fragments 477-819, 820-3069 and 3070-3137 of SEQ ID NO:1, or thecomplementary nucleic acids therto, and which is capable of suppressingcell proliferation.

[0007] The invention further comprises a recombinant polypeptide whichsuppresses cell proliferation and is selected from the group consistingof

[0008] (a) a polynucleotide that is coded by the DNA sequence shown inSEQ ID NO:1.

[0009] (b) a polypeptide that is coded by DNA sequences which hybridizeunder stringent conditions with each of the nucleic acid fragments477-819, 820-3069 and 3070-3137 of SEQ ID NO:1. or the complementary DNAsequences thereto, and a polypeptide that is coded by DNA sequenceswhich, if there was no degeneracy of the genetic code, would hybridizeunder stringent conditions with the sequences defined in a) or b) above.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The polypeptides according to the invention, which are describedimmediately above, can be defined by their DNA sequences as well as bythe amino acid sequence derived therefrom.

[0011] The del-27 polypeptide can occur in natural allelic variationswhich differ from individual to individual. Such variations of the aminoacids are usually amino acid substitutions. However, they may also bedeletions, insertions or additions of amino acids to the total sequence.The del-27 protein according to the invention can be in glycosylated ornon-glycosylated form depending on the cell type in which it isexpressed and the extent to which it is expressed in a particular cell.Polypeptides with tumor-suppressive activity can easily be identified bya tumor growth inhibition assay using carcinoma cells expressing saidpolypeptides and measuring the proliferation capacity and apoptosis inrelation to carcinoma cells not expressing said polypeptides.

Definitions

[0012] As used herein, the following terms have the following meanings:“Polypeptide with del-27 activity or del-27” means also proteins withminor amino acid variations but with substantially the same del-27activity. Substantially the same means that the activities are of thesame biological properties and the polypeptides show preferably at least75% homology in amino acid sequence. More preferably, the amino acidsequences are at least 90% identical.

[0013] The term “nucleic acid molecule” denotes a polynucleotide whichcan be, for example, a DNA, RNA, or derivatized active DNA or RNA. DNAand/or RNA molecules are preferred, however.

[0014] The term “hybridize under stringent conditions” means that twonucleic acid fragments are capable of hybridization to one another understandard hybridization conditions described in Sambrook et al.,“Expression of cloned genes in E. coli” in Molecular Cloning: Alaboratory manual (1989) Cold Spring Harbor Laboratory Press, New York,USA, 9.47-9.62 and 11.45 - 11.61.

[0015] More preferably, “stringent conditions” as used herein refer tohybridization in 6.0×SSC at about 45° C., followed by a wash of 2.0×SSCat 50° C. For selection of the stringency the salt concentration in thewash step can be selected, for example from about 2.0×SSC at 50° C., forlow stringency, to about 0.2×SSC at 50° C., for high stringency. Inaddition, the temperature in the wash step can be increased from lowstringency conditions at room temperatures, about 22° C., to highstringency conditions at about 65° C.

[0016] The term “isolated” as used throughout this application refers toa nucleic acid or polypeptide having del-27 activity and beingsubstantially free of cellular material or culture medium, when producedby recombinant DNA techniques, or being substantially free of chemicalprecursors or other chemicals, when synthesized chemically. An isolatednucleic acid is preferably free of sequences which naturally flank thenucleic acid (i.e. sequences located at the 5′ and the 3′ ends of thenucleic acid) in the organism from which the nucleic acid is derived.

[0017] del-27 can be purified after recombinant production by affinitychromatography using known protein purification techniques, includingimmunoprecipitation, gel filtration, ion exchange chromatography,chromatofocussing, isoelectric focussing, selective precipitation,electrophoresis, and the like.

[0018] The polypeptides according to the invention can be produced byrecombinant means or synthetically. Non-glycosylated del-27 polypeptideis obtained when it is produced recombinantly in prokaryotes. With theaid of the nucleic acid sequences provided by the invention it ispossible to search for the del-27 gene or its variants in genomes of anydesired cells (e.g. apart from human cells, also in cells of othermammals), to identify these and to isolate the desired gene coding forthe del-27 protein. Such processes and suitable hybridization conditionsare known to a person skilled in the art and are described, for examplein Sambrook, J., et al., “Expression of cloned genes in E. coli” inMolecular Cloning: A laboratory manual (1989) Cold Spring HarborLaboratory Press, New York, USA, and B. D. Hames, S. G. Higgins, Nucleicacid hybridisation—a practical approach (1985) IRL Press, Oxford,England. In the current application, the standard protocols described inthese publications were usually used for the experiments reported infra.

[0019] The use of recombinant DNA technology enables the production ofnumerous active del-27 derivatives. Such derivatives can, for example,be modified in individual or several amino acids by substitution,deletion or addition. The derivatization can, for example, be carriedout by means of site directed mutagenesis. Such variations can be easilycarried out by a person skilled in the art (J. Sambrook, B. D. Hames,loc. cit.). It merely has to be ensured by means of the below-mentionedtumor cell growth inhibition assay that the characteristic properties ofdel-27 are preserved. The invention therefore is also directed to del-27polypeptides that are the products of a prokaryotic or eukaryoticexpression of an exogenous DNA.

[0020] With the aid of the disclosed nucleic acids coding for an del-27protein, the proteins according to the invention can be obtained in areproducible manner and in large amounts. For expression in prokaryoticor eukaryotic organisms, such as prokaryotic host cells or eukaryotichost cells, the nucleic acid sequence is integrated into suitableexpression vectors, according to methods familiar to a person skilled inthe art. Such an expression vector preferably contains aregulatable/inducible promoter. These recombinant vectors are thenintroduced for the expression into suitable host cells such as, e.g., E.coli as a prokaryotic host cell or Saccharomyces cerevisiae, Teratocarcinoma cell line PA-I sc 9117 (Büittner et al., Mol. Cell. Biol. 11(1991) 3573-3583), insect cells, CHO or COS cells as eukaryotic hostcells, and the transformed or transduced host cells are cultured underconditions which allow an expression of the heterologous gene. Theisolation of the protein can be carried out according to known methodsfrom the host cell or from the culture supernatant of the host cell.Such methods are described for example by Ausubel I., Frederick M.,Current Protocols in Mol. Biol. (1992), John Wiley and Sons, New York.Also in vitro reactivation of the protein may be necessary if it is notfound in soluble form in the cell culture.

[0021] The invention further comprises recombinant expression vectorswhich are suitable for the expression of del-27 and the above-describedrelated proteins, recombinant host cells transfected with suchexpression vectors, as well as a process for the recombinant productionof a protein which is coded by a tumor suppressor gene del-27.

[0022] The invention further comprises a method for detecting a nucleicacid molecule of tumor suppressor gene del-27, comprising incubating asample (e.g. body fluids such as blood, cell lysates) with the isolatednucleic acid molecule according to the invention and determininghybridization under stringent conditions of said isolated nucleic acidmolecule to a target nucleic acid molecule as a determination ofpresence of a nucleic acid molecule which is the del-27 tumor suppressorgene.

[0023] The invention further comprises a method for producing a proteinwhich is capable of suppressing cell proliferation by expressing anexogenous DNA in prokaryotic or eukaryotic host cells and isolation ofthe desired protein, wherein the DNA

[0024] a) is coded by the DNA sequence shown in SEQ ID NO:1, or

[0025] b) is coded by DNA sequence which hybridizes under stringentconditions with each of the nucleic acid fragments 477-819, 820-3069 and3070-3137 of SEQ ID NO:1, or the complementary DNA sequences, thereto,or

[0026] c) is coded by a DNA sequence which, if there was no degeneracyof the genetic code, would hybridize under stringent conditions with anyof the sequences defined in a) or b) above.

[0027] The invention further comprises an isolated protein according tothe invention which is encoded by a nucleic acid molecule having thenucleotide sequence set forth in SEQ ID NO:1.

[0028] The present invention relates to the cloning and characterizationof a tumor suppressor gene which is especially characterized as a tumorprogression gene. The functional loss of the tumor suppressor geneaccording to the invention (del-27) releases contact inhibition andanchorage dependence in tumor cells. Therefore the loss of del-27 genecorrelates with a more aggressive behavior of the tumor cells and alsothe potential of the formation of metastasis.

[0029] According to the invention, del-27 and its expression productscan be used to inhibit tumor growth, preferably of sporadic tumors (inparticular, lung and bladder carcinomas) in vivo, preferably by somaticgene therapy.

[0030] In combination with retro- or adenoviral vectors the observedtumor suppressive activity of the del-27 cDNA or products of it can beused to inhibt tumor growth of sporadic tumors (in particular incarcinomas such as lung and bladder carcinomas) in vivo by somatic genetherapy.

EXAMPLES

[0031] The following examples, references and sequence listing areprovided to aid the understanding of the present invention, the truescope of which is set forth in the appended claims. It is understoodthat modifications can be made in the procedures set forth withoutdeparting from the spirit of the invention.

Materials and Methods

[0032] Screening and Analysis of Phage Clones:

[0033] Phage pools containing 10⁵ plaque forming units produced fromnormal human genomic libraries (Clontech, Cat.# HL1067J; Stratagene,Cat.# 946205) and human fetal and placental cDNA libraires (Clontech,Cat.# HL1065a and # HL1075b) were screened by the polymerase chainreaction (PCR). PCR conditions were as described in Wieland, I., andBöhm, M., Cancer Res. 54 (1994) 1772-1774. For plaque screening filterlifts were hybridized with 32p-labeled probes and washed under standardconditions as described in Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y. Cold Spring HarborLaboratory, 1989. Postive phage were rescreened by PCR and plaque lifthybridization. Single positive plaques were isolated and phage DNA wasprepared using a kit (Qiagen, Cat.# 12523). Inserts were analysed byrestriction enzyme cleavage and Southern blot hybridization. For genomicclones, subdones of the phage inserts were produced by cleavage withSacI (Boehringer Mannheim) and cloning (Sambrook, J., et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y. Cold SpringHarbor Laboratory, 1989) into the SacI site of plasmid vector pT7T3(Stratagene). Selection of single-copy fragments was based on agarosegel electrophoresis of restriction enzyme cleaved inserts andcounter-selection of repetitive sequences using total genomic DNA as aprobe in Southern blot hybridizations (Seltmann, M., et al., Cytogenet.Cell Genet. 67 (1994) 46-51). Genomic Southern blot hybridizations wereperformed as described (Wieland, I., and Böhm, M., Cancer Res, 54 /1994)1772-1774; Wieland, I., et al., Proc. Natl. Acad. Sci. USA 87 (1992)2720-2724).

DNA Sequencing

[0034] Both strands of overlapping cDNA clones were sequenced by acommerically available source (Eurogentec, Seraing, Bel.). DNA sequenceswere analysed using DNAsis, sequence analysis software (Hitachi).

Cell Lines and Tumor Specimens

[0035] Human lung cancer cell lines were obtained and cultivated asdescribed in Wieland, I., and Böhm, M., Cancer Res, 54 /1994) 1772-1774;Wieland, I., et al., Proc. Natl. Acad. Sci. USA 87 (1992) 2720-2724.

Example 1

[0036] Analysis of the del-27 Genomic Region

[0037] For analysis of the region flanking the del-27 sequence normalhuman genomic phage libraries were screened by PCR for the presence of a356bp PCR product of the del-27 sequence (Wieland, I., and Böhm, M.,Cancer Res, 54 /1994) 1772-1774). Two overlapping phage clones covering21 kb of the del-27 region were isolated and subcloned into a plasmidvector. Single-copy subclones from the del-27 region were used ashybridization probes on genomic Southern blots. Probe p17E contained aninternal HindII site that is hybridized to a 4.3 kb and a second largerHindII fragment. In human lung carcinoma cell line SK-LC-17, however,the 4.3 kb HindII fragment was missing while a smaller fragment of 1.3kb was apparent. Thus, subdone p17E identified a homozygous deletion of3 kb in this cell line. This homozygous deletion includes the previouslyisolated del-27 sequence. A PstI site was also lost resulting in arearranged PstI restriction fragment. Only 3.5 kb apart from thisdeletion a second homozygous deletion of 1.4 kb was identified in thesame cell line using subclone p13B as a probe. Further homozygousdeletions or rearrangements within the 21 kb region were not observed bySouthern blotting experiments. This demonstrates that homozygousdeletions accompanied by genomic rearrangements occurred within 10 kb ofthe del-27 region in human lung carcinoma cell line SK-LC-17. Probe p13Bproduced, in addition to the strong hybridizing signal from the del-27region on chromosome 5, weaker signals on genomic Southern blots. Todetermine whether probe p13B cross-hybridized with another chromosomalregion in the human genome, a somatic cell hybrid panel was analysed(Wieland, I., et al., Proc. Natl. Acad. Sci. USA 87 (1992) 2720-2724).Cross-hybridizing signals were detected in somatic cell hybridscontaining human chromosome 13. This indicates that a sequencehomologous to the p13B sequence from the del-27 region exists on humanchromosome 13. To investigate whether subclones p17E and p13B detectpotential exon sequences, the subclones were analysed for cross-speciesconservation. Both subclones hybridized to genomic fragments fromseveral species. In particular, subdone p13B appeared to be highlyconserved because it hybridized to all mammalian DNAs tested (man,African green monkey, mouse, rat, hamster, dog, cow) and also tochicken. This suggested that exon sequences of a gene exist in thedel-27 region.

Example 2

[0038] Isolation of the del-27 cDNA

[0039] Human cDNA libraries were screened by PCR using primers deducedfrom the highly conserved clone p13B. Several overlapping clones wereisolated and sequenced. The aligned cDNA of 3665bp contains an openreading frame of 2661bp (from nucleotide postion 477 to 3137). Thiscorresponds to an 887 amino acid sequence. Comparison of the del-27 cDNAwith known cDNAs from GeneBank identified a region of homology (with 90%identity) from nucleotide position 820 to 3069 to a mouse cDNA (GenBankaccession number

[0040] U57368, MMU57368) encoding a putative transmembrane proteininvolved in contact inhibition and anchorage dependent growth.

[0041] These results suggest that functional loss of the del-27 geneand/or related genes releases contact inhibition and anchoragedependence in tumor cells. This is supported by the observation thatloss of the del-27 gene correlates with a more aggressive behavior ofthe tumor cells in bladder carcinoma (Böhm, M., et al., Int. J. Cancer72 (1997) 1-5).

Example 3

[0042] Functional test of the del-27 cDNA as a Tumor Suppressor Gene

[0043] The tumor suppressive activity of the del-27 cDNA can be testedby introduction of either the entire coding region or parts of thecoding region of the del-27 cDNA into carcinoma cells:

[0044] The entire coding region or in frame parts of the coding regionare cloned into the tetracycline-inducible expression vector pUHD10-3(Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89 (1992)5547-5551). Together with a selection marker the different constructsare then electroporated into carcinoma cells containing the rtTa-gene onplasmid pUHD172-1neo (Gossen, M., et al., Science 268 (1995) 1766-1769).In selected cell clones expression of the del-27 cDNA is induced bytreatment of the cells with tetracycline or doxycycline. In vitro, tumorsuppressive activity of the del-27 cDNA constructs are determined bymeasuring the proliferative capacity and apoptosis of induced cellscompared to non-induced cells. The generated carcinoma cells are furtherinvestigated in an in vivo mouse model. Cells are injected intoimmune-deficient mice. Expression of the del-27 cDNA constructs in thecarcinoma cells is induced by tetracycline when small tumor nodules areapparent. Tumor suppression (tumor growth inhibition, tumor regression)is monitored in the induced and non-induced state.

1 2 1 3691 DNA Homo sapiens CDS (477)..(3137) 1 gggccggggt gcggagggagtccctgaaac ttctcccaac agccccaggg ccaatcttcc 60 ctcagcccac tcccctcttcgccagctccc aggtccccac tcctgtaccg ggggtggggt 120 ggggagccag gcccgtctcccgctgggaca cacacagggg ccggggagcg gggacgggac 180 ccccgaggcg ggggggacgaaccgacagac agacggctgg gcgcccgccc cagcgggcag 240 gcaggcagga ggaggcggaggcgggggtac gacggggagc actgggtctg gggagtttcc 300 tctcaactat cgggggagaaactccccgca gccggaggaa agacccagac agtgttttcc 360 tcccgggggc cgtgctcccccgccccgcgt agcggcggtc gccgccaccg gcgcctccac 420 ctctaccatc tcctctttctccaccacctc gggccccggt gtccccggcc agcact atg 479 Met 1 ccc atc tta ctgttc ctg ata gac acg tct gcc tct atg aac cag cgc 527 Pro Ile Leu Leu PheLeu Ile Asp Thr Ser Ala Ser Met Asn Gln Arg 5 10 15 agc cat ctg ggc accacc tac ctg gac acg gcc aaa ggc gcg gta gag 575 Ser His Leu Gly Thr ThrTyr Leu Asp Thr Ala Lys Gly Ala Val Glu 20 25 30 acc ttc atg aag ctc cgtgcc cgg gac cct gcc agc aga gga gac agg 623 Thr Phe Met Lys Leu Arg AlaArg Asp Pro Ala Ser Arg Gly Asp Arg 35 40 45 tat atg ctg gtc act ttc gaagag ccg ccc tat gct atc aag gct gga 671 Tyr Met Leu Val Thr Phe Glu GluPro Pro Tyr Ala Ile Lys Ala Gly 50 55 60 65 tgg aaa gaa aac cat gca acgttt atg aat gaa ttg aaa aac ctt cag 719 Trp Lys Glu Asn His Ala Thr PheMet Asn Glu Leu Lys Asn Leu Gln 70 75 80 gct gaa gga ctt acg act ctt ggccaa tcc cta agg aca gct ttt gat 767 Ala Glu Gly Leu Thr Thr Leu Gly GlnSer Leu Arg Thr Ala Phe Asp 85 90 95 tta tta aat tta aat aga tta gta actggc ata gac aac tat ggg cag 815 Leu Leu Asn Leu Asn Arg Leu Val Thr GlyIle Asp Asn Tyr Gly Gln 100 105 110 gga aga aac cct ttt ttc ttg gag ccagca ata att atc aca att act 863 Gly Arg Asn Pro Phe Phe Leu Glu Pro AlaIle Ile Ile Thr Ile Thr 115 120 125 gat ggg agc aag ttg act acc acc agtgga gtc cag gat gag ctt cat 911 Asp Gly Ser Lys Leu Thr Thr Thr Ser GlyVal Gln Asp Glu Leu His 130 135 140 145 tta cct ctt aat tct cct ttg cctgga agt gaa ttg acc aag gaa cct 959 Leu Pro Leu Asn Ser Pro Leu Pro GlySer Glu Leu Thr Lys Glu Pro 150 155 160 ttt cgt tgg gat cag aga ctc tttgca tta gtg ttg cgg ttg cct ggc 1007 Phe Arg Trp Asp Gln Arg Leu Phe AlaLeu Val Leu Arg Leu Pro Gly 165 170 175 acc atg tca gta gaa tca gaa cagttg aca ggt gtg cct tta gat gac 1055 Thr Met Ser Val Glu Ser Glu Gln LeuThr Gly Val Pro Leu Asp Asp 180 185 190 tct gca atc aca cca atg tgt gaagtg aca ggc ggc cgt tca tat tct 1103 Ser Ala Ile Thr Pro Met Cys Glu ValThr Gly Gly Arg Ser Tyr Ser 195 200 205 gtg tgt tct cca aga atg ctt aatcag tgt ctg gag tcc ttg gtg cag 1151 Val Cys Ser Pro Arg Met Leu Asn GlnCys Leu Glu Ser Leu Val Gln 210 215 220 225 aaa gta caa agt ggg gtg gtaata aac ttt gaa aaa gca gga cca gat 1199 Lys Val Gln Ser Gly Val Val IleAsn Phe Glu Lys Ala Gly Pro Asp 230 235 240 cct tcc cct gta gaa gat gggcag cca gat ata tca agg cct ttt gga 1247 Pro Ser Pro Val Glu Asp Gly GlnPro Asp Ile Ser Arg Pro Phe Gly 245 250 255 tct cag cct tgg cat agc tgtcac aaa ctc ata tat gtc aga cca aat 1295 Ser Gln Pro Trp His Ser Cys HisLys Leu Ile Tyr Val Arg Pro Asn 260 265 270 cct aaa act ggg gtt cct ataggt cat tgg cct gtt cca gag tct ttt 1343 Pro Lys Thr Gly Val Pro Ile GlyHis Trp Pro Val Pro Glu Ser Phe 275 280 285 tgg cca gat caa aat tcg ccaaca cta cca cct cgt aca tct cat cct 1391 Trp Pro Asp Gln Asn Ser Pro ThrLeu Pro Pro Arg Thr Ser His Pro 290 295 300 305 gta gtg aag ttt tcc tgtaca gac tgt gaa cca atg gtt att gat aaa 1439 Val Val Lys Phe Ser Cys ThrAsp Cys Glu Pro Met Val Ile Asp Lys 310 315 320 ctt cct ttt gac aaa tatgag ttg gaa cct tca cca ctg act caa ttt 1487 Leu Pro Phe Asp Lys Tyr GluLeu Glu Pro Ser Pro Leu Thr Gln Phe 325 330 335 atc ctg gaa agg aaa tctcct caa aca tgt tgg cag gtg tac gtg agc 1535 Ile Leu Glu Arg Lys Ser ProGln Thr Cys Trp Gln Val Tyr Val Ser 340 345 350 aat agt gca aaa tac agtgaa ctt ggt cat cct ttt ggt tac ttg aaa 1583 Asn Ser Ala Lys Tyr Ser GluLeu Gly His Pro Phe Gly Tyr Leu Lys 355 360 365 gcc agt aca gca ctg aactgt gtc aac tta ttt gtg atg cct tac aat 1631 Ala Ser Thr Ala Leu Asn CysVal Asn Leu Phe Val Met Pro Tyr Asn 370 375 380 385 tat cca gtc ctt cttccc ctc tta gat gac ttg ttt aaa gtg cat aaa 1679 Tyr Pro Val Leu Leu ProLeu Leu Asp Asp Leu Phe Lys Val His Lys 390 395 400 gca aaa cca aca ttgaag tgg aga cag tca ttt gaa agt tat ttg aag 1727 Ala Lys Pro Thr Leu LysTrp Arg Gln Ser Phe Glu Ser Tyr Leu Lys 405 410 415 aca atg cct ccc tactat ctt ggg ccc ttg aag aaa gct gtt agg atg 1775 Thr Met Pro Pro Tyr TyrLeu Gly Pro Leu Lys Lys Ala Val Arg Met 420 425 430 atg gga gca cct aaccta ata gca gac agt atg gaa tat gga ctt agt 1823 Met Gly Ala Pro Asn LeuIle Ala Asp Ser Met Glu Tyr Gly Leu Ser 435 440 445 tac agt gtc att tcatac ctc aaa aaa ctg agt caa cag gcc aaa ata 1871 Tyr Ser Val Ile Ser TyrLeu Lys Lys Leu Ser Gln Gln Ala Lys Ile 450 455 460 465 gaa tct gat cgagtc att gga tct gta ggc aaa aaa gta gta cag gag 1919 Glu Ser Asp Arg ValIle Gly Ser Val Gly Lys Lys Val Val Gln Glu 470 475 480 act gga ata aaagtc cgg agc cga tca cat ggt tta tca atg gca tat 1967 Thr Gly Ile Lys ValArg Ser Arg Ser His Gly Leu Ser Met Ala Tyr 485 490 495 agg aaa gat tttcaa caa ctc ctc cag gga att tca gag gat gtc cct 2015 Arg Lys Asp Phe GlnGln Leu Leu Gln Gly Ile Ser Glu Asp Val Pro 500 505 510 cac aga ctg ctagac ctt aat atg aag gaa tac act ggg ttc caa gtt 2063 His Arg Leu Leu AspLeu Asn Met Lys Glu Tyr Thr Gly Phe Gln Val 515 520 525 gct ttg ctg aataag gat ttg aag cca cag aca ttt aga aat gct tat 2111 Ala Leu Leu Asn LysAsp Leu Lys Pro Gln Thr Phe Arg Asn Ala Tyr 530 535 540 545 gac ata ccaaga cga aat ctt ttg gat cac tta aca aga atg aga tct 2159 Asp Ile Pro ArgArg Asn Leu Leu Asp His Leu Thr Arg Met Arg Ser 550 555 560 aat ctt ttgaag agc act cgc aga ttt ctg aaa gga cag gac gaa gat 2207 Asn Leu Leu LysSer Thr Arg Arg Phe Leu Lys Gly Gln Asp Glu Asp 565 570 575 caa gtg cacagt gtt cct ata gca caa atg ggg aac tac cag gaa tac 2255 Gln Val His SerVal Pro Ile Ala Gln Met Gly Asn Tyr Gln Glu Tyr 580 585 590 ctc aag caagta cct tct cca cta aga gaa ctt gat cct gat cag cca 2303 Leu Lys Gln ValPro Ser Pro Leu Arg Glu Leu Asp Pro Asp Gln Pro 595 600 605 cga agg ttgcat aca ttt ggc aac ccc ttt aag ctg gat aag aag ggt 2351 Arg Arg Leu HisThr Phe Gly Asn Pro Phe Lys Leu Asp Lys Lys Gly 610 615 620 625 atg atgata gat gaa gca gat gaa ttt gtg gct gga cct caa aat aaa 2399 Met Met IleAsp Glu Ala Asp Glu Phe Val Ala Gly Pro Gln Asn Lys 630 635 640 cat aaacga ccc gga gaa cca aat atg caa ggg atc cct aaa aga cgt 2447 His Lys ArgPro Gly Glu Pro Asn Met Gln Gly Ile Pro Lys Arg Arg 645 650 655 cgg tgtatg tct cca cta cta aga ggc aga cag cag aat cct gtt gta 2495 Arg Cys MetSer Pro Leu Leu Arg Gly Arg Gln Gln Asn Pro Val Val 660 665 670 aac aatcat att ggg gga aaa gga cca cct gca cct aca act caa gca 2543 Asn Asn HisIle Gly Gly Lys Gly Pro Pro Ala Pro Thr Thr Gln Ala 675 680 685 cag ccagat ctt att aaa cct ctt cct ctt cat aaa att tca gaa acc 2591 Gln Pro AspLeu Ile Lys Pro Leu Pro Leu His Lys Ile Ser Glu Thr 690 695 700 705 actaat gat tcg ata ata cat gat gtg gtt gaa aat cat gtt gca gac 2639 Thr AsnAsp Ser Ile Ile His Asp Val Val Glu Asn His Val Ala Asp 710 715 720 caactt tca tca gac att aca cca aat gct atg gat acg gaa ttt tca 2687 Gln LeuSer Ser Asp Ile Thr Pro Asn Ala Met Asp Thr Glu Phe Ser 725 730 735 gcatct tct cca gcc agt tta ctg gaa cgg cca acc aat cat atg gag 2735 Ala SerSer Pro Ala Ser Leu Leu Glu Arg Pro Thr Asn His Met Glu 740 745 750 gctctt ggt cat gac cat tta gga acc aat gac ctc act gtt ggt gga 2783 Ala LeuGly His Asp His Leu Gly Thr Asn Asp Leu Thr Val Gly Gly 755 760 765 ttttta gaa aat cat gag gag cca aga gat aaa gaa caa tgt gct gaa 2831 Phe LeuGlu Asn His Glu Glu Pro Arg Asp Lys Glu Gln Cys Ala Glu 770 775 780 785gag aac ata cca gca tct tca ctc aac aaa gga aag aaa ttg atg cat 2879 GluAsn Ile Pro Ala Ser Ser Leu Asn Lys Gly Lys Lys Leu Met His 790 795 800tgc aga agc cat gaa gag gtc aat act gaa cta aaa gca caa ata atg 2927 CysArg Ser His Glu Glu Val Asn Thr Glu Leu Lys Ala Gln Ile Met 805 810 815aaa gag atc cga aag cca gga aga aaa tat gaa aga atc ttc act tta 2975 LysGlu Ile Arg Lys Pro Gly Arg Lys Tyr Glu Arg Ile Phe Thr Leu 820 825 830ctg aag cat gtg caa ggc agt tta caa aca aga cta ata ttt tta caa 3023 LeuLys His Val Gln Gly Ser Leu Gln Thr Arg Leu Ile Phe Leu Gln 835 840 845aat gtc att aaa gaa gca tca agg ttt aaa aaa cga atg cta ata gaa 3071 AsnVal Ile Lys Glu Ala Ser Arg Phe Lys Lys Arg Met Leu Ile Glu 850 855 860865 caa ctg gag aac ttc ttg gat gaa att cat cga aga gcc aat cag atc 3119Gln Leu Glu Asn Phe Leu Asp Glu Ile His Arg Arg Ala Asn Gln Ile 870 875880 aac cat att aat agc aat taaaagaaaa tagaatgtgg ccacttattt 3167 AsnHis Ile Asn Ser Asn 885 cactatcttc ttcaaataca aagtaaataa caagactgttgtgatcttgc attcattttc 3227 tgacatgcat tgttggctat ttgaaatact aaaagcaaatctacagatcc tttttccatc 3287 attttacagt gaccttttct tcattttggt ttatttttgtaatgtgaaaa gtatcactct 3347 aaaaaacatt tttaatttaa caaactaaaa atattcctccaaatctcttg cttgtcattg 3407 actcttgcgt gtcaatttcc ctcaggttct attttcttaaaccaaccttt aaaattgtca 3467 cctctgttaa ggttgaactt tgccaaaaaa aaaagaagttactttgtaat ttttggggaa 3527 aaaagcacat acattaaaac taggtaatgt tttgtatatacagttatttt ggatatatta 3587 ttgtaagttg tacaaatgta ttttgaagaa tatttaagaaaagcactttt gttattccat 3647 caataaatgc tctattttaa aaaaaaaaaa aaaaaaaaaaaaaa 3691 2 887 PRT Homo sapiens 2 Met Pro Ile Leu Leu Phe Leu Ile AspThr Ser Ala Ser Met Asn Gln 1 5 10 15 Arg Ser His Leu Gly Thr Thr TyrLeu Asp Thr Ala Lys Gly Ala Val 20 25 30 Glu Thr Phe Met Lys Leu Arg AlaArg Asp Pro Ala Ser Arg Gly Asp 35 40 45 Arg Tyr Met Leu Val Thr Phe GluGlu Pro Pro Tyr Ala Ile Lys Ala 50 55 60 Gly Trp Lys Glu Asn His Ala ThrPhe Met Asn Glu Leu Lys Asn Leu 65 70 75 80 Gln Ala Glu Gly Leu Thr ThrLeu Gly Gln Ser Leu Arg Thr Ala Phe 85 90 95 Asp Leu Leu Asn Leu Asn ArgLeu Val Thr Gly Ile Asp Asn Tyr Gly 100 105 110 Gln Gly Arg Asn Pro PhePhe Leu Glu Pro Ala Ile Ile Ile Thr Ile 115 120 125 Thr Asp Gly Ser LysLeu Thr Thr Thr Ser Gly Val Gln Asp Glu Leu 130 135 140 His Leu Pro LeuAsn Ser Pro Leu Pro Gly Ser Glu Leu Thr Lys Glu 145 150 155 160 Pro PheArg Trp Asp Gln Arg Leu Phe Ala Leu Val Leu Arg Leu Pro 165 170 175 GlyThr Met Ser Val Glu Ser Glu Gln Leu Thr Gly Val Pro Leu Asp 180 185 190Asp Ser Ala Ile Thr Pro Met Cys Glu Val Thr Gly Gly Arg Ser Tyr 195 200205 Ser Val Cys Ser Pro Arg Met Leu Asn Gln Cys Leu Glu Ser Leu Val 210215 220 Gln Lys Val Gln Ser Gly Val Val Ile Asn Phe Glu Lys Ala Gly Pro225 230 235 240 Asp Pro Ser Pro Val Glu Asp Gly Gln Pro Asp Ile Ser ArgPro Phe 245 250 255 Gly Ser Gln Pro Trp His Ser Cys His Lys Leu Ile TyrVal Arg Pro 260 265 270 Asn Pro Lys Thr Gly Val Pro Ile Gly His Trp ProVal Pro Glu Ser 275 280 285 Phe Trp Pro Asp Gln Asn Ser Pro Thr Leu ProPro Arg Thr Ser His 290 295 300 Pro Val Val Lys Phe Ser Cys Thr Asp CysGlu Pro Met Val Ile Asp 305 310 315 320 Lys Leu Pro Phe Asp Lys Tyr GluLeu Glu Pro Ser Pro Leu Thr Gln 325 330 335 Phe Ile Leu Glu Arg Lys SerPro Gln Thr Cys Trp Gln Val Tyr Val 340 345 350 Ser Asn Ser Ala Lys TyrSer Glu Leu Gly His Pro Phe Gly Tyr Leu 355 360 365 Lys Ala Ser Thr AlaLeu Asn Cys Val Asn Leu Phe Val Met Pro Tyr 370 375 380 Asn Tyr Pro ValLeu Leu Pro Leu Leu Asp Asp Leu Phe Lys Val His 385 390 395 400 Lys AlaLys Pro Thr Leu Lys Trp Arg Gln Ser Phe Glu Ser Tyr Leu 405 410 415 LysThr Met Pro Pro Tyr Tyr Leu Gly Pro Leu Lys Lys Ala Val Arg 420 425 430Met Met Gly Ala Pro Asn Leu Ile Ala Asp Ser Met Glu Tyr Gly Leu 435 440445 Ser Tyr Ser Val Ile Ser Tyr Leu Lys Lys Leu Ser Gln Gln Ala Lys 450455 460 Ile Glu Ser Asp Arg Val Ile Gly Ser Val Gly Lys Lys Val Val Gln465 470 475 480 Glu Thr Gly Ile Lys Val Arg Ser Arg Ser His Gly Leu SerMet Ala 485 490 495 Tyr Arg Lys Asp Phe Gln Gln Leu Leu Gln Gly Ile SerGlu Asp Val 500 505 510 Pro His Arg Leu Leu Asp Leu Asn Met Lys Glu TyrThr Gly Phe Gln 515 520 525 Val Ala Leu Leu Asn Lys Asp Leu Lys Pro GlnThr Phe Arg Asn Ala 530 535 540 Tyr Asp Ile Pro Arg Arg Asn Leu Leu AspHis Leu Thr Arg Met Arg 545 550 555 560 Ser Asn Leu Leu Lys Ser Thr ArgArg Phe Leu Lys Gly Gln Asp Glu 565 570 575 Asp Gln Val His Ser Val ProIle Ala Gln Met Gly Asn Tyr Gln Glu 580 585 590 Tyr Leu Lys Gln Val ProSer Pro Leu Arg Glu Leu Asp Pro Asp Gln 595 600 605 Pro Arg Arg Leu HisThr Phe Gly Asn Pro Phe Lys Leu Asp Lys Lys 610 615 620 Gly Met Met IleAsp Glu Ala Asp Glu Phe Val Ala Gly Pro Gln Asn 625 630 635 640 Lys HisLys Arg Pro Gly Glu Pro Asn Met Gln Gly Ile Pro Lys Arg 645 650 655 ArgArg Cys Met Ser Pro Leu Leu Arg Gly Arg Gln Gln Asn Pro Val 660 665 670Val Asn Asn His Ile Gly Gly Lys Gly Pro Pro Ala Pro Thr Thr Gln 675 680685 Ala Gln Pro Asp Leu Ile Lys Pro Leu Pro Leu His Lys Ile Ser Glu 690695 700 Thr Thr Asn Asp Ser Ile Ile His Asp Val Val Glu Asn His Val Ala705 710 715 720 Asp Gln Leu Ser Ser Asp Ile Thr Pro Asn Ala Met Asp ThrGlu Phe 725 730 735 Ser Ala Ser Ser Pro Ala Ser Leu Leu Glu Arg Pro ThrAsn His Met 740 745 750 Glu Ala Leu Gly His Asp His Leu Gly Thr Asn AspLeu Thr Val Gly 755 760 765 Gly Phe Leu Glu Asn His Glu Glu Pro Arg AspLys Glu Gln Cys Ala 770 775 780 Glu Glu Asn Ile Pro Ala Ser Ser Leu AsnLys Gly Lys Lys Leu Met 785 790 795 800 His Cys Arg Ser His Glu Glu ValAsn Thr Glu Leu Lys Ala Gln Ile 805 810 815 Met Lys Glu Ile Arg Lys ProGly Arg Lys Tyr Glu Arg Ile Phe Thr 820 825 830 Leu Leu Lys His Val GlnGly Ser Leu Gln Thr Arg Leu Ile Phe Leu 835 840 845 Gln Asn Val Ile LysGlu Ala Ser Arg Phe Lys Lys Arg Met Leu Ile 850 855 860 Glu Gln Leu GluAsn Phe Leu Asp Glu Ile His Arg Arg Ala Asn Gln 865 870 875 880 Ile AsnHis Ile Asn Ser Asn 885

1. An isolated nucleic acid molecule having the sequence SEQ ID NO:1. 2.An isolated nucleic acid molecule of claim 1 having the sequence 477-819of SEQ ID No:1.
 3. An isolated nucleic acid molecule of claim 1 havingthe sequence 820 to 3069 of SEQ ID NO:1.
 4. An isolated nucleic acidmolecule of claim 1 having sequence the 3070 to 3137 of SEQ ID NO:1. 5.An isolated polypeptide having the sequence SEQ ID NO:2.
 6. An isolatedpolypeptide encoded by DNA sequence SEQ ID NO:1.
 7. An isolatedpolypeptide of claim 6 encoded by DNA sequence 477-819 of SEQ ID NO:1.8. An isolated polypeptide of claim 6 encoded by DNA sequence 820 to3069 of SEQ ID NO:1.
 9. An isolated polypeptide of claim 6 encoded byDNA sequence the 3070 to 3137 of SEQ ID NO:1.
 10. A method fordetermining that a tumor cell lacks metastatic potential by detectingthe presence of tumor suppressor gene del-27 in the tumor cell,comprising: a. contacting a sample containing tumor cell DNA with anucleic acid probe having the sequence SEQ ID NO:1 under stringenthybridization conditions; and b. detecting a tumor cell DNA capable ofhybridizing to the full length of the probe, thereby determining thatthe tumor cell lacks metastatic potential.
 11. A method of determiningthat a tumor cell has metastatic potential by detecting the deletion orrearrangement of tumor suppressor gene del-27 in the tumor cell,comprising: a. contacting a sample containing tumor cell DNA with anucleic acid probe having the sequence SEQ ID NO:1 under stringenthybridization conditions; and b. detecting no tumor cell DNA capable ofhybridizing to the full length of the probe, thereby determining thatthe tumor cell has metastatic potential.